pMBP-LbCas12a vector (V001402) Gene synthesis in pMBP-LbCas12a backbone

COA reports Sequencing Results MSDS Download GenBank File(.gb)

Price Information

Cat No.	Plasmid Name	Availability	Buy one, get one free! (?)
V001402	pMBP-LbCas12a	In stock, instant shipping

Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Basic Vector Information

The pMBP-LbCas12a plasmid is designed for high-yield expression and purification of the Lachnospiraceae bacteriumCas12a protein. It utilizes an MBP (maltose-binding protein) fusion tag to improve soluble protein yield and a His-tag for purification via nickel affinity chromatography. This vector is widely used to produce Cas12a for CRISPR-based genome editing and diagnostic applications.

Vector Name:: pMBP-LbCas12a

Antibiotic Resistance:: Ampicillin, 100 μg/mL

Length:: 9653 bp

Type:: Bacterial Expression

Replication origin:: ori

Source/Author:: Jennifer Doudna

Copy Number:: High Copy

Promoter:: tet

Cloning Method:: Gibson Cloning

5' Primer:: GATGAAGCCCTGAAAGACGCGCAG

3' Primer:: CTA GTT ATT GCT CAG CGG T

Growth Strain(s):: DH5alpha

Growth Temperature:: 37℃

pMBP-LbCas12a vector Map

Copy Sequence View Map

Plasmid Protocol

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.

3. Close the tube and incubate for 10 minutes at room temperature.

4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.

5. Store the plasmid at -20 ℃.

6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it

General Plasmid Transform Protocol

1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.

2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.

3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.

4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.

5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.

References

Chen JS, Ma E, Harrington LB, Da Costa M, Tian X, Palefsky JM, Doudna JA. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science. 2018 Apr 27;360(6387):436-439. doi: 10.1126/science.aar6245. Epub 2018 Feb 15. Erratum in: Science. 2021 Feb 19;371(6531):eabh0317. doi: 10.1126/science.abh0317. PMID: 29449511; PMCID: PMC6628903.

Misiurina MA, Chirinskaite AV, Fotina AS, Zelinsky AA, Sopova JV, Leonova EI. New PAM Improves the Single-Base Specificity of crRNA-Guided LbCas12a Nuclease. Life (Basel). 2022 Nov 18;12(11):1927. doi: 10.3390/life12111927. PMID: 36431062; PMCID: PMC9698171.

Sun D, Zhu Y, Wang M, Wang J, Cheng W, Li Z, Deng Y, Ou X, Jia R, Chen S, Zhu D, Liu M, Zhao X, Yang Q, Wu Y, Zhang S, Huang J, He Y, Wu Z, Cheng A. A RT-ERA-CRISPR/Cas12a assay for rapid point-of-care duck hepatitis A virus detection. Poult Sci. 2025 Aug;104(8):105316. doi: 10.1016/j.psj.2025.105316. Epub 2025 May 23. PMID: 40449106; PMCID: PMC12166872.

pMBP-LbCas12a vector Sequence

LOCUS       40924_29901        9653 bp DNA     circular SYN 08-JUN-2021
DEFINITION  Expresses 10xHis-MBP fused to LbCas12a.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9653)
  AUTHORS   Chen JS, Ma E, Harrington LB, Da Costa M, Tian X, Palefsky JM, 
            Doudna JA
  TITLE     CRISPR-Cas12a target binding unleashes indiscriminate 
            single-stranded DNase activity.
  JOURNAL   Science. 2018 Apr 27;360(6387):436-439. doi: 
            10.1126/science.aar6245. Epub 2018 Feb 15.
  PUBMED    29449511
REFERENCE   2  (bases 1 to 9653)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 9653)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1126/science.aar6245"; journalName: "Science"; date: 
            "2018-04-27- 27"; volume: "360"; issue: "6387"; pages: "436-439"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9653
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     990..1009
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI,
                     reverse primer"
     promoter        1093..1111
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1200..1222
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1242..1268
                     /codon_start=1
                     /label=9xHis
                     /note="9xHis affinity tag"
                     /translation="HHHHHHHHH"
     CDS             1281..2381
                     /codon_start=1
                     /label=MBP
                     /note="maltose binding protein from E. coli"
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     CDS             2436..2456
                     /codon_start=1
                     /label=TEV site
                     /note="tobacco etch virus (TEV) protease recognition and 
                     cleavage site"
                     /translation="ENLYFQS"
     CDS             2463..6146
                     /codon_start=1
                     /label=LbCpf1
                     /note="CRISPR-associated protein from Lachnospiraceae
                     bacterium ND2006 (Zetsche et al., 2015)"
                     /translation="MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAE
                     DYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKE
                     IAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFS
                     EEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEG
                     EFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVL
                     SDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKN
                     GPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQ
                     LQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKD
                     LLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYS
                     KDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNG
                     NYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKL
                     IDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLV
                     EEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKK
                     EELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKI
                     NTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTD
                     YHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSG
                     FKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQ
                     NGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALD
                     YKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQ
                     QGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRN
                     YEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTS
                     VKH"
     terminator      6289..6336
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(6702..6730)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein
                     gene"
     primer_bind     complement(6757..6775)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     promoter        6843..6947
                     /label=AmpR promoter
     CDS             6948..7805
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      7979..8567
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8721..8738
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    complement(8753..8892)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     8978..9000
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(8997..9185)
                     /codon_start=1
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
                     /translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"